Inamine et al. (Gene 73:175-183) identified an operon in M. pneumoniae that encodes a gene responsible for attachment of M. pneumoniae to the human host. The PI gene product appears to be essential for attachment, although the role that the other genes in this operon play in attachment remain unknown. In this project we will focus on Orf 4, and its possible role in attachment. This project has two primary goals: 1) The DNA encoding this Orf will be subjected to site-specific mutagenesis to allow for the expression of this gene in E. coli and 2) the expressed protein will be purified from E. coli for use in various biological experiments. M. pneumoniae does not read the genetic code in the same manner as most bacteria, it uses the TGA codon to encode tryptophan, rather than using it as a translation termination signal. In order to express this gene, the TGA codons found in this Orf need Id to TGG. A PCR-based site-directed mutagenesis procedure will be used. Individual regions will be amplified using the PCR, and various restriction sites will be incorporated into the Orf. All of the fragments will be cloned into pMal-p-2. This cloning vector is specially designed to facilitate the purification of cloned proteins. The purified protein will be studied in a variety of ways. One hypothesis that we have is that this protein is involved in the proteolytic processing of the protein encoded by Orf4 of the PI operon. We will use the purified protein to determine if the Orf1 protein can cleave the Orf4 protein. This project will interrelate with another project from this lab "Cloning and characterization of the protease..". We will use substrates purified in that project to determine if the Orf1 protein encodes a protease. We will use a variety of animal seras to determine if the Orf 1protein is immunogenic in animals. We will generate antibodies against the purified protein and determine where this protein is localized in the mycoplasma cell.